This invention relates to immobilization of receptors on a support in a liquid chromatographic system.
The combinatorial synthesis of chemical libraries has created an enormous pool of possible new drug candidates. Indeed, synthetic capabilities have outstripped the ability to determine corresponding biological activity. An initial step in the resolution of this problem has been the development of microtiter plates which contain immobilized receptors/antibodies. The use of these plates can rapidly reduce the number of possible candidates in a combinatorial pool from thousands to hundreds. However, assignment of relative activity within the reduced pool of compounds remains a slow and repetitive process.
The relationship between basic pharmacological processes and liquid chromatographic (LC) studies have been emphasized by the inclusion of biomolecules as active components of chromatographic systems. A wide variety of immobilized biopolymer-based LC stationary phases (BP-SPs) have been developed using proteins, enzymes, cellulose and amylose, macrocyclic antibodies and liposomes. Indeed, it has been demonstrated that the chromatographic retention and selectivity of BP-SPs are related to the properties of the non-immobilized biopolymer. For example, retention of a compound on an SP column containing immobilized human serum albumin has been used to evaluate the binding properties of the compounds to proteins.
This invention provides a immobilized receptors on supports in liquid chromatographic systems. Using the methods of the invention, it is possible to immobilize receptors on supports, then expose those receptors to agents that might attach to the receptors. It is then possible to expose the supports with the receptors, followed by liquid chromatographic studies to determine whether attachment of the agent to the receptor has occurred. It is, of course, also possible to expose the receptors on the supports to substances that might inhibit interaction between the agent that is known to interact with the receptor, then expose the supports with the receptors to the agent to determine whether or not the proposed inhibitor will, in fact, inhibit attachment to the receptor. Hence, using means of the invention, it is possible to test interaction of potential drugs and receptors and also to evaluate possible agents for inhibition of receptor interactions.
It is the purpose of this invention to provide means for immobilization of membrane receptors in order to study relative a ligand-receptor interactions utilizing LC techniques. This method has been tested using neuronal nicotinic acetylcholine receptors as an example. The fundamental processes of drug action, absorption, distribution and receptor activation, are dynamic in nature and have much in common with the basic mechanisms involved in chromatographic distribution. Indeed, the same basic intermolecular interactions (hydrophobic, electrostatic and hydrogen bonding) determine the behavior of chemical compounds in both biological and chromatographic environments.
Although membrane receptors play an important role in drug activity and are key targets in combinatorial screens, they have not been included in LC systems. This has been due, in part, to the disruption of the tertiary structure of the receptor produced by covalent immobilization on a solid LC support. One solution to this problem is the immobilization of membrane receptors in the phospholipid monolayer of an immobilized artificial membrane (IAM) LC stationary phase.
The method provides means of evaluating the attachment of agents to receptors comprising the steps of:
(a) immobilizing receptors on artificial membrane supports in a column,
(b) exposing the supports with the receptors to test agents at varying concentrations in a liquid chromatographic system,
(c) eluting the test agent from the column, and
(d) evaluating the elution profile of the test materials from the column.
Using this method, it is possible to evaluate the interaction of the test agent with the receptor. Following elution, it is possible to directly determine molecular structure by passing the elute through other testing devices such as a mass spectrometer.
In the examples, the IAM LC stationary phase is derived from the covalent immobilization of 1-myristoyl-2-[(13-carboxyl)tridecanoyl]-sn-3-glycerophosphocholine on aminopropyl silica and resembles xc2xd of a cellular membrane. In the IAM phase, the phosphatidylcholine headgroups form the surface of the support and the hydrocarbon side chains produce a hydrophobic interphase which extends from the charged headgroup to the surface of the silica. (This phase has been previously used to immobilize hydrolytic and cofactor-dependent enzymes without loss of activity.)
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels formed from five homologous subunits oriented like barrel staves center pore. The nAChRs are the primary excitatory neurotransmitter receptors on skeletal muscles and autonomic ganglia in the peripheral nervous system of vertebrates. In the central nervous system, the nAChRs play important roles in modulating functions of other neurotransmitters. Sixteen different subunits of nAChRs have been identified so far. These subunits combine to form a variety of nAChR subtypes. A stably transfected mammalian cell line established recently expresses a single subtype of rat nAchRs, xcex13/xcex24, at a level that is at least 30 times higher than the expression level of native nAChRs in mammalian tissue. The rat xcex13xcex24 nAChR, therefore, was selected for the initial development of a receptor-bearing LC stationary phase.
The xcex13/xcex24 nAChRs prepared from the cell line were solubilized using a detergent solution. The resulting detergent-receptor solution was mixed with the dry IAM LC support and then dialyzed against Tris-HCl buffer [50 mM, pH 7.4]. Additional buffer was added to the IAM support and the mixture was vortexed, centrifuged and the supernatant decanted. The resulting IAM LC support contained approximately 60 mg protein per gram IAM support.
The ability of the immobilized receptors to bind known nAChR ligands was determined using a [3H]epibatidine binding assay protocol designed for cell membrane homogenates. In the binding assays, the xcex13/xcex24 IAM support suspension showed 98% specific binding with 5 xcex7M active receptor per gram immobilized protein, comparable to 100% specific binding with 8.6 xcex7M per gram protein found in parallel experiments for the receptor-detergent solution. No specific binding was found to the native IAM LC support.
Materials and Methods
Immobilization of Receptor on the Chromatopraphic Matrices
The cultured cells in which rat xcex13/xcex24 subtype of neural nicotinic acetylcholine receptor (nAChR) were expressed by transfected cell line KXxcex13xcex24R2 were harvested in 30 ml of Tris-HCl buffer (50 mM, pH 7.4) and homogenized for 20 minutes with a Brinkman Polytron homogenizer. The homogenates were centrifuged at 35,000xc3x97g for 10 minutes and the supernatant was discarded. The resulting pellets were suspended in 6 ml of 2% cholate in Tris-HCl buffer (50 mM, pH 7.4) and stirred for two hours in an ice bath. After centrifugation at 35,000xc3x97g, the supernatant was collected for the immobilization. IAM packing materials (200 mg) were washed three times by centrifugation with Tris-HCl buffer (50 mM, pH 7.4). The IAM materials were suspended in 4 ml of receptor-detergent solution, stirred for one hour at room temperature and dialyzed against 2xc3x971 L buffer for 24 hours at 4xc2x0 C. When detergents were depleted from the mixture, the receptors were hydrophobically entrapped in the phospholipid monolayer on the surface of IAM materials. The receptor-IAM materials were washed with buffer by centrifugation at 4000xc3x97g.
Frontal Chromatography Using Immobilized NR(xcex14xcex22)-IAM
The NR(xcex14xcex22)-IAM packing materials were packed in an HR glass column (Pharmacia Biotech) and connected with an HPLC pump. 15-20 ml of [3H]-EB in buffer or in the same buffer containing drugs was applied by a 25 ml sample superloop (Pharmacia Biotech, Uppsala, Sweden) and run at a flow rate of 0.4 ml/min at room temperature. The elution profile was monitored by an on-line radioactivity HPLC detector.